Impact of divergence of residual feed intake on triglyceride metabolism-related gene expression in meat-type ducks

Triglyceride (TG) metabolism is a key factor that affects residual feed intake (RFI); however, few studies have been conducted on the related gene expression in poultry. The aim of the present study was to investigate the expression of genes and their associations with RFI in meat-type ducks. Weight gain and feed intake (FI) at an age 21–42 days were measured and the RFI was calculated. Quantitative PCR was used to test the expression of the six identified genes, namely peroxisome proliferator activated receptor γ (PPARγ), glycerol kinase 2 (GK2), glycerol-3-phosphate dehydrogenase 1 (GPD1), glycerol kinase (GYK), lipase E (LIPE), and lipoprotein lipase (LPL) in the duodenum in the high RFI (HRFI) and low RFI (LRFI) groups. The results demonstrated that daily feed intake, feed conversion ratio (FCR), and RFI were markedly higher in HRFI ducks than those in LRFI ducks. Moreover, the levels of expression of PPARγ, GK2, and LIPE were significantly higher in the LRFI group than those in the HRFI group. Correlation analysis showed that PPARγ, GK2, and LIPE were significantly negatively associated with FCR and RFI. Furthermore, gene expression levels were negatively associated with the measured phenotype. The association of GK2 with PPARγ, GPD1, LPL, and LIPE was positive. The relationship between the TG related gene and RFI was further verified to potentially develop pedigree poultry breeding programs. The results of this study suggested that the expression of genes correlated with TG metabolism and transport is up-regulated in the duodenum of ducks with high feed efficiency. PPARγ, GK2, and LIPE are important genes that affect RFI. The results of the present study provide information that could facilitate further explorations of the mechanism of RFI and potential markers at the molecular and cellular levels.


Introduction
The poultry industry in China is well developed and plays a pivotal role in the agricultural economy. As poultry, ducks receive the largest amount of feed in China and have been an important part of the livestock production. In modern breeding, feed cost accounts for approximately 70% of the total raising cost [1]. To increase the benefit of meat-type duck breeding, research is currently focusing on improving feed efficiency (FE). FE is evaluated based on feed conversion ratio (FCR) and residual feed intake (RFI) [2]. Kennedy [3] proposed the RFI value, which is defined as the difference between the actual and expected feed intake (FI) based on the weight gain and average animal weight. As an evaluation index of FE for livestock and poultry, RFI is based on weight difference, independent of production [4], and has medium heritability [5]. RFI can be selected for application in FE improvement, whereas FCR is a ratio trait with limited breeding applications. Therefore, as RFI is a useful assessment for FE in meat-type duck breeding, researchers are committed to finding the potential genetic markers of RFI [1,6].
Lipid metabolism is a key factor affecting RFI. Different levels of energy are consumed when depositing lean tissue and fat of an equivalent weight. The energy metabolism of low RFI (LRFI) animals is reflected in improvements in nutrition and energy distribution efficiency for growth and muscle deposition [7]. In poultry, LRFI broilers had lower abdominal fat rates and higher breast muscle rates than HRFI broilers [8], and both abdominal fat and breast muscle rates were strikingly correlated with the breeding value of RFI traits [9]. As a product of lipolysis, triglyceride (TG) decomposition and synthesis is an essential part of lipid metabolism. The total amount of fat deposition and types of fatty acids are the products of the comprehensive regulation of TG synthesis and metabolism in the body, which are regulated at different levels [10]. Based on previous studies, we selected six key genes involved in triglyceride synthesis and catabolism. GYK [11], GK2 [12], and GPD1 [13] are involved in the synthesis of TG, while PPARγ [14], LIPE [15], and LPL [16] are key genes involved in the catabolism of TG. The duodenum is an important site for the decomposition and utilization of TG [17]. The activity of pancreatic lipase in the intestine can affect the absorption of fat in the body [18]. However, few studies have compared the expression characteristics of genes related to TG metabolism in the duodenum of different meat-type ducks.
Therefore, in the present study, we evaluated the complete genealogical records of the Qiangying duck Breeding Co. Ltd., China using quantitative PCR to explore the TG metabolismrelated gene expression differences in HRFI and LRFI and its relevance to RFI. We provide a theoretical basis for the improved breeding of meat-type ducks and selection of high FE ducks to reveal the control of the biological mechanisms associated with RFI.

Ethics statement
All protocols including animal experiments were carried out in adherence to the regulations and guidelines established by the Administration of Affairs Concerning Experimental Animals (Ministry of Science and Technology, China, revised in June 2004). All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Anhui Agricultural University (SYXK 2016-007). The animals in the study had ad libitum access to water and feed and were humanely euthanized by cervical dislocation. All efforts were made to relieve the suffering of the ducks. until 21 days of age. At 21 days, all ducks were weighed, and a total of 1000 meat-type male ducks with similar body weight (1042.1 ± 87.2 g) were placed in the individual cages (55 × 50 × 40 cm) for feeding until 42 days of age. All ducks were exposed to continuous illumination (24 L: 0 D) for the first three days after hatching, followed by a 20 L: 4 D lighting regime until the end of the experiment. All ducks were housed in the same house and fed the same basal diet at room temperature. During the experiment, the feeding management and immunization procedures were carried out according to uniform standards, and all the experimental ducks were free to feed and water ad libitum. Ingredient composition and nutrient levels of the basal diet used in the present study are shown in Table 1.

Trait measurement
We recorded the 21-day-old body weight (BW21), 42-day-old body weight (BW42), FI, body weight gain (BWG), and calculated the average daily feed intake (ADFI) and average daily gain (ADG), FCR, and RFI [19,20], using the following formula of RFI: where RFI is the residual feed intake; ADFI is the daily feed intake; MBW 0.75 is the metabolic body weight; ADG is the average daily gain; b 0 is the intercept, and b 1 and b 2 are regression coefficients. The linear fitting function in SAS version 9.4 software (SAS Institute, Cary, NC, USA) was used to calculate the RFI value of the meat-type ducks.

Sample collection and total RNA extraction
At 42 days of age, the RFI of all individuals was determined. According to the results of the RFI ranking, the experimental duck population was divided into HRFI and LRFI groups, and considering their FCR values, eight meat-type male ducks were selected each group. After the ducks were slaughtered, the duodenum was collected and immediately stored in a 2.0 mL RNA containing centrifuge tube at 4˚C overnight and then stored at −80˚C until further analyses. Total RNA from duodenal epithelial tissues was extracted using a total RNA kit (Omega Bio-Tek, Doraville, GA, USA). The RNA concentration and purity of samples were determined using NanoDrop spectrophotometer (Thermo Fisher Scientific, New York, NY, USA) in 1.0% agarose gel electrophoresis. SuperMix was synthesized using cDNA for qPCR (Yeasen, Shanghai, China) for the reverse transcription of the isolated total RNA. All experimental procedures were carried out as per the manufacturer's protocols.

Primers, complementary DNA synthesis, and quantitative PCR analysis
According to the duck genome sequence published in the GenBank, the primers of six genes were designed using Primer 5.0 software (Premier Biosoft International, Palo Alto, CA, USA), as shown in Table 2. Hydroxymethylbilane synthase (HMBS) was employed as an internal reference gene, and quantitative PCR was performed using the Hieff qPCR SYBR Green Master Mix kit (Yeasen, Shanghai, China) with the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) according to manufacturer's instructions. Thermal cycling parameters were as follows: 95˚C for 5 min, followed by 40 cycles of 10 s at 95˚C, 20 s at 60˚C, and 20 s at 72˚C, and a final step of 5 min at 72˚C. The reaction specificity was confirmed by melt curve analysis. The relative transcriptional levels of the various genes were confirmed using the 2 -ΔΔCt (in which Ct is the cycle threshold period) method.

Statistical analysis
The 2 −ΔΔCt method was used to calculate the relative gene expression levels according to the Ct values of the target and internal genes [21]. Experiments were based on using a completely

Feed efficiency traits
FE trait data are presented in Table 3, showing that ADFI, FCR, and RFI were significantly lower (P < 0.01) in LRFI ducks than those in HRFI ducks. However, there was no significant difference in ADG and MBW 0.75 between the HRFI and LRFI groups (P > 0.05).

Gene expression
The relative levels of expression of TG metabolism-related genes in the duodenums of HRFI and LRFI groups are displayed in Fig 1. The expression levels of PPARγ (P < 0.05), GK2 (P < 0.01), and LIPE (P < 0.01) in the LRFI group were markedly higher than those in the HRFI group. There was no statistical difference in GYK, GPD1, and LPL between the two RFI groups (P > 0.05).

Relationship between gene expression and feed efficiency traits
As shown in Table 4, the relative expression levels of PPARγ were significantly and negatively correlated with MBW 0.75 (P < 0.05), FCR (P < 0.05), RFI (P < 0.05), and ADFI (P < 0.01). Similarly, the relative levels of expression of GK2 were negatively correlated with MBW 0.75 (P < 0.05), RFI (P < 0.05), ADFI (P < 0.01), and FCR (P < 0.01). The relative levels of expression of LIPE were significantly negatively correlated with ADFI, FCR, and RFI (P < 0.01). It can be concluded that the relative levels of expression of PPARγ, GK2, and LIPE were negatively correlated with ADFI, FCR, and RFI in the meat-type ducks.

Correlation between gene expression and triglyceride metabolism
The results of the correlation analysis based on relative gene expression are shown in Table 5. The expression of TG metabolism-related genes showed a positive correlation, of which, PPARγ gene expression was significantly positively correlated with the expression of GK2, GPD1, LPL, and LIPE (P < 0.01). GYK expression was positively correlated with GPD1 expression (P < 0.05). GK2 expression was significantly positively correlated with GPD1, LPL, and LIPE expression (P < 0.01). GPD1 expression was significantly positively correlated with LPL and LIPE expression (P < 0.01). LPL expression was positively correlated with LIPE expression (P < 0.05).

Discussion
Enhancing the FE of livestock and poultry could reduce breeding costs, increase the economic benefits of breeding, and reduce environmental pollution [22]. In the present study, the ADFI, FCR, and RFI of meat-type ducks were remarkably different between HRFI and LRFI groups (P < 0.01). The results are in agreement with the findings of previous studies on chickens [8] and mule ducks [23]. These studies indicate that poultry with LRFI has higher FE and lower breeding costs. Zhang et al. [1] reported that the sebum rate of Peking ducks was remarkably positively correlated with RFI. Individuals with LRFI have lower fat content and lower expression of various proteins related to fat deposition synthesis such as fatty acid synthase, fatty acid binding protein, and peripherin. Zhuo et al. [8] found that chicken abdominal fat that differentially expressed genes were mainly enriched in lipid metabolism, coagulation, and immune regulatory pathways. The duodenum is an important organ for nutrient digestion and absorption. Most of the fat in the duodenum is absorbed into intestinal cells by enzymes, and then synthesized and stored in the body. Feed intake is an important factor affecting RFI [7], and dietary fat is mostly consumed in the form of TG. Previous transcriptome studies of the duodenum of chicken showed that RFI was related to the regulation of nutrient digestion and absorption and that intestinal health can improve FE. Ten genes related to digestive health and function were also identified as candidate genes for RFI [6].
Glycerol metabolism has a major effect on the catabolism of fatty acids and sugars [24]. The PPAR signaling pathway plays crucial roles in lipid metabolism, adipogenesis, and insulin sensitivity. PPARγ belongs to the nuclear receptor superfamily of ligand-activated transcription factors (TFs) [25,26]. It is highly expressed in adipocytes and is a key regulator of adipocyte differentiation, insulin signaling, fat energy conversion, bone morphogenesis and differentiation, and immune response [27][28][29]. It plays a pivotal role in lipid metabolism and related diseases, such as obesity [30], diabetes [31], atherosclerosis [32], and cancer [33]. PPARγ can control lipid metabolism and monounsaturated fatty acid synthesis [14] and regulate TG synthesis in mammary epithelial cells of goats [34] and cows [35]. Increased PPARγ expression in adipose tissue has also been observed in low FE mice [36]. Moreover, long-term feeding with the PPARγ activator activates PPARγ and improves FI and FE in rats, accompanied by high leptin levels, when compared to rats in other high-fat feeding groups [37]. Chicken PPARγ is co-expressed in adipocytes with other adipogenesis-related TFs and regulates the expression of adipogenesis-related genes [38]. However, the expression of PPARγ in the duodenum is different from that in adipose tissue. Ojano-Dirain et al. [39] found that PPARγ expression in the duodenum of chickens with high FE was higher than that of chickens with low FE, which is consistent with the results of our study. At the same time, Kelly et al. [40] found that PPARγ expression was negatively correlated with FCR and RFI, which was also consistent with the results of our study. This may be because, during fat accumulation, PPARγ can also promote TG to release fatty acids, ensure intercellular fatty acid transport, and promote fatty acid esterification by activating fatty acid binding proteins and lipoprotein esterase [26]. Since avian lipogenesis mainly occurs in the liver, and the duodenum is the place for nutrient digestion and absorption, the expression of PPARγ in the duodenum of HRFI meat-type ducks is distinctly lower than that of LRFI meat-type ducks. This study found that the expression level of PPARγ was negatively associated with ADFI, MBW 0.75 , FCR, and RFI, and the expression level of PPARγ in the duodenum of LRFI ducks was distinctly higher than that of HRFI. The phenotypic results were consistent with the differential expression of PPARγ in LRFI and HRFI. Upstream regions of LPL and fatty acid binding protein 4 (FABP4) promoters can bind to PPAR, after which LPL and FABP4 are activated and expressed [41], which is consistent with the significant positive relationship between PPARγ and LPL expression levels observed in this study. LIPE is an important member of the TG lipase family that mediates adipocyte lipolysis [42]. It is one of the key enzymes affecting fat deposition in animals. Greenberg et al. [43] first found this protein in epididymal adipocytes of mice. LIPE, as the rate limiting enzyme of TG hydrolysis, is responsible for converting TG into diacylglycerol [44,45]. At the same time, the activity of this enzyme is controlled by hormones, which is also known as hormone sensitive lipase (HSL). Moreover, LIPE can not only catalyze the release of fatty acids from TG stored in adipocytes [15], but also catalyze the release of cholesterol from cholesterol esters in steroid tissues for steroid hormone synthesis [46]. The expression level of LIPE affects marbling and intramuscular fat (IMF) content, which is a potential molecular marker that affects bovine IMF, and it can be applied in breeding selection [45]. Liu et al. [47] used transcriptome analysis of differential expression of lipid metabolism-related genes in Shandong black and Luxi cattle and found that the LIPE is the key node of various metabolic pathways, and its relative expression is higher in cattle breeds with higher IMF content. The difference in the expression of LIPE may augment fatty acid related metabolic pathways of beef cattle and positively mediate polyunsaturated fatty acids, stearic acids, and linoleic acids. In a study of lipid metabolism in piglets, when low density lipoprotein (LDL) and cholesterol levels in the blood decreased and high density lipoprotein (HDL) increased, the expression of LIPE and LPL decreased significantly [48], in agreement with the gene expression observed in the present study. In our study, the expression of LIPE in meat-type ducks in the HRFI group was markedly higher than that in the LRFI group (P < 0.01), and LIPE was markedly positively associated with ADFI and FCR, which may be because LIPE is the rate limiting enzyme for TG hydrolysis. In addition, there was a positive relationship between the gene expression of LIPE and that of LPL.
GYK and GK2 are the enzymes responsible for converting glycerol into glycerol 3-phosphate (G3P), the substrate for glycolysis and lipid synthesis [11,12,24], as glycerol levels are elevated in the absence of GK [49]. Considering that glycerol levels usually reflect the state of fat mobilization, Assis et al. [50] found that glucocorticoids decrease thermogenic capacity and increase TG synthesis by GK activation in the brown adipose tissue of rats. In our study, GK2 was negatively correlated with MBW 0.75 , RFI, ADFI, and FCR. In addition, the level of expression of GK2 in the LRFI group was markedly higher than that in the HRFI group. The change in the gene expression level may affect the TG metabolism of HRFI and LRFI meat-type ducks, and then in turn affect the lipid metabolism process of the body, changing the RFI.

Conclusion
In conclusion, our findings indicated that the expression of PPARγ, GK2, and LIPE involved in TG metabolism and transport was significantly up-regulated in the duodenum of meat-type ducks with high feed efficiency. Moreover, association analysis indicated that the relative expression levels of PPARγ, GK2, and LIPE were significantly and negatively correlated with FCR, RFI, and ADFI. Additionally, there was a significant positive correlation between the relative expression of PPARγ, GK2, and LIPE genes.